Protein Quality of Alfalfa Protein Concentrates Obtained by Freezing

J. Agric. Food Chem. 1997, 45, 797−802 797 Protein Quality of Alfalfa Protein Concentrates Obtained by Freezing Teresa Hernańdez,*,† Carmen Martıńez,† Amelia Hernańdez,† and Gloria Urbano‡ Departamento de Nutricioń y Bromatologıá, Universidad de Alcala´ de Henares, 28871 Alcala´ de Henares, Madrid, Spain, and Departamento de Fisiologıá, Universidad de Granada, 18071 Granada, Spain Freezing protein curd was prepared from alfalfa juice and extracted with 2-propanol, either unwashed or after washing with water. To examine the effect of extraction on digestibility, in vitro digestibility was analyzed for the two extracted protein concentrates and the unextracted protein concentrate. Two methods, one direct, the pH-stat method, and one indirect, the dietary fiber method, were used to determine in vitro digestibility. The protein quality of the unextracted freezing concentrate was also determined by bioassays. The in vivo digestibility of this concentrate (82.8) was lower than that of soybean meal, while the biological value (75.4) was similar. Thus, the lower net protein utilization (62.4) of this concentrate as compared to soybean meal can be ascribed to its lower digestibility. Extraction of the concentrate with 2-propanol lowered in vitro digestibility about 11 and 2% by pH-stat and dietary fiber methods, respectively, while washing the concentrate with water before extraction either had no effect on in vitro digestibility or lowered it slightly. There was an inversely proportional relationship statistically significative between the content of dietary and insoluble fiber in the concentrates and their in vitro digestibilities. Keywords: Protein quality; protein concentrates; alfalfa INTRODUCTION 1983). Another method involves extracting whole protein concentrates using organic solvents; this method Unfractionated alfalfa leaf protein concentrates (LPCs) gives higher yields, and the extracted concentrate may have been regarded as a potential alternative to con- be stored for extended periods without need of low ventional sources of edible protein. LPCs have been temperature or exclusion of oxygen (Bray, 1977). assessed as food for children (Kamalanathan et al., 1969, Extraction of the freezing curd with 2-propanol yields 1970; Oke, 1971, 1973; Olatunbosum et al., 1972; concentrates with a high protein content, minimum Kamalanathan and Devadas, 1975; Pirie, 1984) and lipid, sugar, and polyphenol contents, and color and included in food formulations (Toosy and Shah, 1974; texture similar to those of white protein concentrates Meimbam et al., 1982; Lencioni et al., 1984, 1987, 1989; (Hernańdez et al., 1988, 1991; Hernańdez and Hernań- Barbeau and Kinsella, 1987; Maciejewicz-Rys and Hanc- dez, 1994). However, for solvent extraction to be a zakowski, 1990). viable method of preparing chlorophyll-free protein Unfractionated LPC can be prepared by treatment concentrates, it is essential to determine how this using heat (Bickoff et al., 1975), acid (Satake et al., process affects the protein quality of the concentrates 1984), organic solvents (Brown et al., 1975), or polyelec- so produced. trolytes (Anelli et al., 1977; Baraniak et al., 1989). The object of the present study was therefore to Unfractionated LPC can also be obtained by freezing. determine the protein quality of alfalfa protein concen- Freezing alfalfa juice produces a curd, called freezing trates prepared either by extracting freezing curd, curd, which contains 50% of the dry matter and 60% of unwashed or washed with water, with 2-propanol or by the nitrogen present in the original juice (Hernańdez directly freeze-drying the freezing curd, and hence to et al., 1988). The nitrogen content in the freezing assess the effect of freezing and extraction on the protein concentrate prepared by freeze-drying the curd is the quality of such concentrates. same as in the unfractionated concentrate. This method of preparation offers advantages, in that it requires MATERIALS AND METHODS neither heat nor the addition of any chemical sub- Concentrate Preparation. Preparation of the freezing stances. However, the effect of freezing on the protein curd has been described elsewhere (Hernańdez et al., 1988, quality of the concentrate so obtained has not yet been 1991). Briefly, alfalfa was harvested, pulped, and pressed. The assessed. juice was poured into small containers and frozen at -25 °C To be suitable for human consumption, LPCs must until use. Each sample was thawed at room temperature for be chlorophyll-free. Such concentrates may be prepared 18 h before use, as needed. Freezing curd so formed was separated from the thawed juice by centrifugation, and some using two different methods. One method is to separate of the freezing curd was freeze-dried to produce freezing the soluble white proteins from the insoluble colored concentrate (FC). Extraction with 2-propanol was carried out protein matter, though this method presents the draw- in a Soxhlet apparatus at the solvent’s boiling point using back of producing lower yields of white protein concen- either untreated curd immediately after preparation, yielding trate (de Fremery et al., 1973; Fiorentini and Galoppini, 2-propanol-extracted freezing concentrate (IFC), or curd after it had been washed with distilled water by centrifugation, yielding water-washed 2-propanol-extracted freezing concen- * Author to whom correspondence should be ad- trate (WIFC) (Figure 1). Following removal of the residual dressed (fax 34-1-8854783). solvent, the extracted FCs were ready for analysis. † Universidad de Alcala´ de Henares. In Vivo Tests of Protein Quality. Apparent (AD) and ‡ Universidad de Granada. true (TD) digestibilities, biological value (BV), and net protein S0021-8561(96)00347-0 CCC: $14.00 © 1997 American Chemical Society 798 J. Agric. Food Chem., Vol. 45, No. 3, 1997 Hernańdez et al. Figure 1. Obtention process of freezing protein concentrates. utilization (NPU) of the FC were determined in rats using the 37 °C, and the pH was adjusted to 8.0 and held there for 10 Thomas-Mitchell balance method, as modified by Eggum min. An automatic titration was then run using the pH-stat (1973). The daily weight gain with respect to daily protein procedure, adding 1 mL of the enzyme solution (end point 7.98, intake (DWG/DPI) was calculated for the balance period. 0.100 M NaOH titration solution). Enzyme activity was In experiments, albino Wistar rats (50-60 g) were housed determined on the basis of the amount of 0.100 M NaOH in individual metabolic cages kept in a thermoregulated room required to keep the pH at 7.98 for exactly 10 min. Percentage (21 ( 1 °C) with a controlled 12 h light/dark period. true digestibility (%TD) of the caseinate was calculated by The rats were divided randomly into two groups of 10 animals each one (5 males, 5 females). One group of rats was %TD ) 76.14 + 47.77B used to determine maintenance requirements. The other group was used in the test diet assay. where B ) mL of added 0.100 M NaOH. Values for sodium To determine the maintenance requirements, the animals caseinate digestibility should fall in the range of 98-102% were fed a low-protein (4%) diet of a highly digestible protein (McDonough et al., 1990a). Otherwise, a new three-enzyme (casein + 5% DL-methionine), a sufficient amount to record solution needs to be made up. depletion (McDonough et al., 1990b). To determine the digestibility of the concentrates, an The FC was the only protein source in the test diet at the amount of sample containing 10 mg of N was transferred to a level of 12% of dry matter (DM). The diets also contained 4% titration cell in 10 mL of distilled water. Once the temperature fat, 8% fiber, 1% vitamin mix, and 4% mineral mix, the last had been stabilized at 37 °C, the pH was adjusted to 8 and two according to AIN (1977). A starch and sugar (1:1) mixture held there for 10 min. An amount of 1 mL of the enzyme completed the diets. solution was then added, and the titration was started (end The diets and water were given ad libitum. The rats were point pH 7.98, 0.100 M NaOH titrant solution). The amount fed the low-nitrogen maintenance diet for 6 days (a 3 day of 0.100 M NaOH required to keep the pH at 7.98 for exactly adaptation period and a 3 day balance period) and the test 10 min was used to determine an uncorrected protein digest- diet for 10 days (a 3 day adaptation period and a 7 day balance ibility (%UTD) value by employing the equation set out above. period). At the end of the study the rats were weighed and The %UTD values were corrected using the value obtained the fecal N, urinary N, and food consumed were determined. for the sodium caseinate solution (McDonough et al., 1990a): The AD, TD, BV, and NPU values of the FC were calculated. Moisture was determined by drying at 105 °C and the total %TD ) (%UTD/%TD caseinate) 100 nitrogen according to the Kjeldahl method using a protein × conversion factor of 6.25. The baseline rate of titrant uptake was determined in runs In Vitro Digestibility Method. The in vitro digestibility in which no enzyme solution was added. After the concentrate of the three alfalfa protein concentrates was determined by samples had been adjusted to pH 8.0, the baseline uptake of two methods, a direct assay and an indirect assay. titrant was measured for 10 min. The digestibility values The direct method used was the pH-stat method developed corrected for the baseline consumption of alkali (%TDb) were by Pedersen and Eggum (1983). A three-enzyme solution was obtained. made up to contain 23 100 units of trypsin (EC 3.4.21.4) A Titrino Model SM 702 automatic titrator equipped with (porcine pancreatic trypsin, type IX, Sigma T-0134), 186 units a 10 mL exchange unit (Metrohm) and a thermostated titration of chymotrypsin (EC 3.4.21.1) (bovine pancreatic chymotrypsin, cell was used for all of the pH-stat titrations.

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